The overall objective is to elucidate the biochemical and subcellular mechanism of action of ryanodine, a drug which depresses contractile force in heart. Since ryanodine apparently alters intracellular Ca2 ion metabolism, we intend to study the effects of ryanodine on basic cellular processes which regulate ionized Ca2 ion concentration in cardiac tissues. These studies will be conducted in vitro with purified preparations of cardiac sarcolemma and sarcoplasmic reticulum. Our preliminary data suggest that the specific site of ryanodine action in heart may be sarcoplasmic reticulum, where the drug acts to increase Ca2 ion storage. We will test directly whether ryanodine blocks Ca2 ion efflux from sarcoplasmic reticulum vesicles, and thereby increases the net uptake of Ca2 ion during active transport conditions. Also, we will characterize the differential stimulation of ryanodine on Ca2 ion uptake by distinct subpopulations of cardiac sarcoplasmic reticulum vesicles. Finally, possible biochemical effects of ryanodine on the enzymes of cardiac sarcolemmal vesicles will be measured. If ryanodine is shown to depress contractility by blocking Ca2 ion efflux channels in sarcoplasmic reticulum, the usefulness of this drug as a powerful and specific molecular probe will be established. As a result of these studies, knowledge will be gained on basic mechanisms of excitation-contraction coupling in heart.